The overarching goal of the proposal is to identify molecules that govern development of the enteric nervous system (ENS) which, if abnormal, may contribute to the pathogenesis of functional and other disorders of Gl motility/secretion. Because the ENS can uniquely regulate the behavior of the gut in the absence of CNS input, and is also large, complex, and structurally/chemically similar to the brain, ENS development is likely to be more "brain-like" than similar to that of other peripheral ganglia. Recently reported molecular defects in the gut of patients with irritable bowel syndrome are compatible with the idea that ENS abnormalities contribute to the pathogenesis of functional disturbances of Gl motility/secretion. If so, the defects would have to be far subtler than those which give rise to an aganglionosis;e.g.: Hirschsprung's disease (HSCR). HSCR-associated mutations affect genes required early in ENS development. We now propose to investigate genes and molecules involved in the later developmental events (about which little is currently known) that could underlie more limited ENS defects, including enteric neurogenesis, synaptogenesis, and the establishment of the extrinsic innervation. To do so, we will use gain- and loss-of-function strategies to identify roles played in ENS ontogeny by the "proneural" basic helix-loop-helix transcription factor, Hand2, the synaptic adhesion molecules, neurexins and neuroligins, and the guidance molecules, netrins, Slit proteins and their respective receptors, deleted in colorectal cancer (DCC) and Robo. Preliminary data that suggest that Hand2 is required for the differentiation of at least subsets of enteric neurons will be confirmed. We will also determine whether Hand2 is necessary for gliogenesis and whether the newly discovered developmental regulation of the subcellular distribution of Hand2 allows it to play a role in neuronal adaptation in mature life. Neurexins and neuroligins are expressed in the developing human and rodent ENS;we will now determine whether their interaction is necessary and sufficient for enteric synapse formation and we will investigate their alternative splicing as a trans-synaptic signaling code. Preliminary observations suggest that netrins guide DCC-expressing vagal sensory axons to the gut. We will extend these studies to the guidance of vagal motor and sacral motor and sensory nerves and determine the roles played by laminin-1 and/or Slit proteins in limiting netrin-mediated attraction.